We use state-of-the-art technologies and computational analyses to understand, at single cell resolution, how the organization of epithelial differentiation is altered by selective perturbations or inflammatory perturbations of te terminal ileum. During the continuous homeostatic renewal process, an epithelial progenitor cell progressively differentiates according to a set of rules set forth by a composite of signaling pathways that are spatially arranged along the crypt-villus axis. Decades of intestinal biology research have established that differentiation proceeds stepwise, with branched decisions resulting in a hierarchical organization of the different functional lineages. How individual cells coherently integrate multiple signaling pathways into a hierarchical differentiation program, and how this program is coordinately altered when perturbed in states of disease are undefined. In inflammatory bowel disease (IBD), the organized intestinal microenvironment is infiltrated by a variety of immune cells that provide additional signaling inputs to epithelial cells. We hypothesize that normal signaling organization is perturbed by these additional inputs, which in turn, either rearranges or abolishes the hierarchical organization of epithelial differentiation, leading to imprecise differentiation, failed maintenance of the mature state, and altered proportions of epithelial cell types. We will coordinate two emerging technologies, MultiOmyx microscopy and Cytometry Time-of-Flight (CyTOF), to interrogate multiple signaling activities and cell differentiation pathways at single cell resolution, in order to use data-driven computational approaches to model differentiation hierarchies. In the two aims of this proposal, we will apply our approaches to study (Aim 1) the effects of specific pathway inhibition, which we hypothesize to maintain hierarchical differentiation, and (Aim 2) the effects of chronic inflammation, which we hypothesize to convert the strict differentiation hierarchy into a plastic landscape. The study of stromal and immune cell contributions to the organization of differentiation will be studied using a combination of enteroid cultures, live imaging, and multiplex microscopy of tissue sections. Future directions include the direct application of our experimental-computational platforms to human patient samples of Crohn's ileitis. Through our findings, we aim to make significant contributions to the understanding, diagnosis and treatment of IBD. The outcomes from this research will hopefully be a gateway towards the study of other complex diseases with undefined etiologies, molecular distribution of many risk factors over many components, and interactions between multiple heterologous systems.